recA Sequences Research Articles

Streptomyces apocyni sp. nov., an endogenous actinomycete isolated from Apocynum venetum.

A novel actinomycete, designated strain TRM 66233T, was isolated from Apocynum venetum L. collected from the Xinjiang Uygur Autonomous Region of China and characterized using a polyphasic taxonomic approach. Phylogenetic analysis based on 16S rRNA gene sequences affiliated strain TRM 66233T with the genus Streptomyces. Strain TRM 66233T showed a high similarity value to Streptomyces bikiniensis NRRL B-1049T (98.07 %) based on the 16S rRNA gene phylogenetic tree. The whole-cell sugar pattern of TRM 66233T consisted of glucose, galactose, mannose and ribose. The predominant menaquinones were MK-9(H2), MK-9(H6), MK-9(H8) and MK-9(H10). The polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol and four unidentified lipids. The major fatty acids were iso-C15 : 0, anteiso-C15 : 0, iso-C16 : 0, C16 : 0 and iso-C17 : 0. The G+C content of the DNA was 70.35 mol%. The DNA-DNA relatedness and average nucleotide identity values as well as evolutionary distances based on multilocus (atpD, gyrB, recA, rpoB and trpB) sequences between strain TRM 66233T and closely related type strains were significantly lower than the recommended threshold values. The whole-genome average nucleotide identity and digital DNA-DNA hybridization values between strain TRM 66233T and S. bikiniensis NRRL B-1049T were 78.86 and 23.2 %, respectively. On the basis of evidence from this polyphasic study, strain TRM 66233T should represent a novel species of the genus Streptomyces, for which the name Streptomyces apocyni sp. nov. is proposed. The type strain is TRM 66233T (=CCTCC AA 2019056T=LMG 31559T).

First report of tobacco bacterial hollow stalk caused by Dickeya chrysanthemi (syn. Erwinia chrysanthemi) in Nanping, China.

Tobacco, Nicotiana tabacum L., family Solanaceae, is grown for its leaves (mature period). It originated in the Mesoamericas but today is cultivated globally. China produces about one-third of the world´s supply of tobacco leaves and is the largest tobacco producer in the world (Hu et al. 2007). In the early summer of 2019, after tobacco has been topped, typical tobacco hollow stalk disease symptoms were observed in two to five percent of the fields in Shaowu and Jianyang counties of Nanping, Fujian province, China. Symptoms consisted of lesions extending from the point of topping downward, resulting in a black hollow stem (harvest period). The symptoms were similar to tobacco hollow stalk disease caused by Pectobacterium species. Four infected stems of different tobacco plants were collected from Nanping (N 27°37'48″, E 118°2'24″) for isolation. Small sections from the stems were surface sterilized with 1% sodium hypochlorite for 3 min, rinsed three times in sterile distilled water (SDW), then macerated in 400 μl of SDW. The suspensions were then streaked on Nutrient agar (NA) and incubated at 28°C in darkness. After 2 days, two predominant isolates that produced circular, convex, small (< 1 mm) colonies without pigmentation, were purified. The BIOLOG GEN III Microplates system was then used to identify the putative pathogenic bacteria based on the results of the biochemical assays read by the plate reader and its associated software and database. After culturing for 48 and 60 h,BIOLOG analysis identified the two isolates as Dickeya chrysanthemi (SIM=0.516, DIST=7.224 and SIM=0.589, DIST=5.984). To confirm its identification, the 16S rDNA gene from strain NPEc1 was amplified with the general primer 27F/1492R (Frank et al. 2008), A BLASTn search in GenBank of this sequence (1,465 bp, Accession No. MN640862) showed over 99% identity to the 16S rDNA of D. chrysanthemi (CP 001655). Partial sequences of housekeeping gene DNA polymerase III, subunits gamma and tau (dnaX), and the recombinase A (recA) gene were also analyzed according to Racix et al. (2014). The 511-bp dnaX sequence (MT597420) and 736-bp recA sequence (MT597421) respectively matched 98.22% and 96.19% with D. chrysanthemi (syn. Erwinia chrysanthemi) strain CFBP 2048 (JX434939) and ICMP 5703 (DQ859873). For pathogenicity testig, six tobacco seedlings of the 45-day-old cultivar Yunyan 87 were planted in nutriculture at room temperature. After three days, approximately 20 μl of each bacterial suspension (107 CFU/ml) of isolates NPEc1 and NPEc2 was injected into the leaf axils of six tobacco stems. As a control, similar seedlings were inoculated with SDW. Twelve hours after inoculation, symptoms of water-soaked decay appeared in the injected leaf axils. After 2 days, these symptoms developed into a severe rot similar to that of naturally infected plants. In contrast, the controls were symptomless. The bacterium was isolated from the rotten tissues and demonstrated a similar identity to the inoculants by the Biolog automated microbial identification system comparison, thus fulfilling Koch's postulates. The inoculation was repeated twice. On the basis of morphological, Biolog Gen III, molecular characteristics and pathogenicity, the causal agent of a hollow stalk rot on tobacco at Nanping, was identified as D. chrysanthemi. Previously, this pathogen was reported on tobacco in Cuba by Pérez (1981) and more recently isolated from poinsettia stems in China (Rungnapha et al. 2008). To our knowledge, this is the first report of Dickeya chrysanthemi causing a hollow stalk disease of toacco in China. Reference cultures have been deposited in CGMCC (NO. MT211276). Further spread of the pathogen may pose a threat to tobacco production in Nanping and other regions in China.

Rhizobium ruizarguesonis sp. nov., isolated from nodules of Pisum sativum L

Four strains, coded as UPM1132, UPM1133T, UPM1134 and UPM1135, and isolated from nodules of Pisum sativum plants grown on Ni-rich soils were characterised through a polyphasic taxonomy approach. Their 16S rRNA gene sequences were identical and showed 100% similarity with their closest phylogenetic neighbors, the species included in the ‘R. leguminosarum group’: R. laguerreae FB206T, R. leguminosarum USDA 2370T, R. anhuiense CCBAU 23252T, R. sophoreae CCBAU 03386T, R. acidisoli FH13T and R. hidalgonense FH14T, and 99.6% sequence similarity with R. esperanzae CNPSo 668T. The analysis of combined housekeeping genes recA, atpD and glnII sequences showed similarities of 92-95% with the closest relatives. Whole genome average nucleotide identity (ANI) values were 97.5-99.7% ANIb similarity among the four strains, and less than 92.4% with closely related species, while digital DNA-DNA hybridization average values (dDDH) were 82-85% within our strains and 34-52% with closely related species. Major fatty acids in strain UPM1133T were C18:1 ω7c / C18:1 ω6c in summed feature 8, C14:0 3OH/ C16:1 iso I in summed feature 2 and C18:0. Colonies were small to medium, pearl-white coloured in YMA at 28°C and growth was observed in the ranges 8-34°C, pH 5.5-7.5 and 0-0.7% (w/v) NaCl. The DNA G+C content was 60.8mol %. The combined genotypic, phenotypic and chemotaxonomic data support the classification of strains UPM1132, UPM1133T, UPM1134 and UPM1135 into a novel species of Rhizobium, for which the name Rhizobium ruizarguesonis sp. nov. is proposed. The type strain is UPM1133T (=CECT 9542T=LMG 30526T).

Taxonomic note: speciation within the operational group Bacillus amyloliquefaciens based on comparative phylogenies of housekeeping genes

Many of the publically available Bacillus 16S rRNA genes and genomes in the NCBI database are inconsistently assigned as B. amyloliquefaciens. The highly conserved nature of the 16S rRNA gene makes it fail to differentiate species within the operational group B. amyloliquefaciens. Here, comparative phylogenies of the complete 16S rRNA, gyrB, rpoB, trpB, recA, and cheA nucleotide sequences of bacterial strains within the operational group were analyzed. As the result, the gyrB, rpoB, and trpB phylogenetic analyses showed stable topology that comprised three monophyletic clades: (i) B. amyloliquefaciens; (ii) B. siamensis; and (iii) B. velezensis. Phylogenies derived by comparison of the gyrB, rpoB, trpB, recA, and cheA with the 16S rRNA gene-derived phylogeny was significant as evaluated by the likelihood ratio test. The trpB, rpoB, and trpB gene-derived phylogenies provide a tool for speciation within the operational group B. amyloliquefaciens.

The ACC-Deaminase Producing Bacterium Variovorax sp. CT7.15 as a Tool for Improving Calicotome villosa Nodulation and Growth in Arid Regions of Tunisia.

Calicotome villosa is a spontaneous Mediterranean legume that can be a good candidate as pioneer plants to limit regression of vegetation cover and loss of biodiversity in Tunisian arid soils. In order to grow legumes in such soils, pairing rhizobia and nodule associated bacteria (NAB) might provide numerous advantages. In this work, cultivable biodiversity of rhizobial symbionts and NAB in nodules of C. villosa plants growing in five arid regions of south Tunisia was characterized. Phylogenetic analysis using 16S rDNA gene, dnak, recA and nodD sequences separated nodule-forming bacteria in six clades associated to genera Ensifer, Neorhizobium, Phyllobacterium and Rhizobium. Among NAB, the strain Variovorax sp. CT7.15 was selected due to its capacity to solubilise phosphate and, more interestingly, its high level of aminocyclopropane-1-carboxylate deaminase (ACC deaminase) activity. C. villosa plants were inoculated with representative rhizobia of each phylogenetic group and co-inoculated with the same rhizobia and strain CT7.15. Compared with single rhizobia inoculation, co-inoculation significantly improved plant growth and nodulation, ameliorated plant physiological state and increased nitrogen content in the plants, independently of the rhizobia used. These results support the benefits of pairing rhizobia and selected NAB to promote legume growth in arid or degraded soils.

Streptomyces cahuitamycinicus sp. nov., isolated from desert soil and reclassification of Streptomyces galilaeus as a later heterotypic synonym of Streptomyces bobili.

A novel actinobacterial strain, designated 13K301T, was isolated from a soil sample collected from the Karakum Desert, Turkmenistan. The taxonomic position of strain 13K301T was revealed by using a polyphasic approach. On the basis of 16S rRNA gene sequence analysis, strain 13K301T belongs to the genus Streptomyces and had highest sequence similarity to 'Streptomyces qaidamensis' S10T (99.2 %), Streptomyces flavovariabilis NRRL B-16367T (98.9 %) and Streptomyces phaeoluteigriseus DSM 41896T (98.8 %), but the strain formed a distinct clade in the phylogenetic tree. The DNA-DNA relatedness and average nucleotide identity values as well as evolutionary distances based on multilocus (atpD, gyrB, recA, rpoB and trpB) sequences between strain 13K301T and closely related type strains were significantly lower than the recommended threshold values. The cell wall contained ll-diaminopimelic acid and the whole-cell hydrolysates were glucose and ribose. Diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol and phosphatidylinositol were determined as the predominant polar lipids. The major menaquinones were identified as MK-9(H8) and MK-9(H6). On the basis of these genotypic and phenotypic data, it is proposed that strain 13K301T should be classified as representative of a novel species of the genus Streptomyces, for which the name Streptomyces cahuitamycinicus sp. nov. is proposed. The type strain is 13K301T (=DSM 106873T=KCTC 49110T). In addition, the whole genome-based comparisons as well as the multilocus sequence analysis revealed that the type strains of Streptomyces galilaeus and Streptomyces bobili belong to a single species. It is, therefore, proposed that S. galilaeus be recognised as a heterotypic synonym of S. bobili for which an emended description is given.

Bradyrhizobium ivorense sp. nov. as a potential local bioinoculant for Cajanus cajan cultures in Côte d'Ivoire.

For many smallholder farmers of Sub-Saharan Africa, pigeonpea (Cajanus cajan) is an important crop to make ends meet. To ascertain the taxonomic status of pigeonpea isolates of Côte d’Ivoire previously identified as bradyrhizobia, a polyphasic approach was applied to strains CI-1BT, CI-14A, CI-19D and CI-41S. Phylogeny of 16S ribosomal RNA (rRNA) genes placed these nodule isolates in a separate lineage from current species of the B. elkanii super clade. In phylogenetic analyses of single and concatenated partial dnaK, glnII, gyrB, recA and rpoB sequences, the C. cajan isolates again formed a separate lineage, with strain CI-1BT sharing the highest sequence similarity (95.2 %) with B. tropiciagri SEMIA 6148T. Comparative genomic analyses corroborated the novel species status, with 86 % ANIb and 89 % ANIm as the highest average nucleotide identity (ANI) values with B. elkanii USDA 76T. Although CI-1BT, CI-14A, CI-19D and CI-41S shared similar phenotypic and metabolic properties, growth of CI-41S was slower in/on various media. Symbiotic efficacy varied significantly between isolates, with CI-1BT and CI-41S scoring on the C. cajan ‘Light-Brown’ landrace as the most and least proficient bacteria, respectively. Also proficient on Vigna radiata (mung bean), Vigna unguiculata (cowpea, niébé) and additional C. cajan cultivars, CI-1BT represents a potential bioinoculant adapted to local soil conditions and capable of fostering the growth of diverse legume crops in Côte d'Ivoire. Given the data presented here, we propose the 19 C. cajan isolates to belong to a novel species called Bradyrhizobium ivorense sp. nov., with CI-1BT (=CCOS 1862T=CCMM B1296T) as a type strain.

Characterization and phylogenetic diversity of Allorhizobium vitis isolated from grapevine in Morocco.

Crown gall, a phytobacteriosis characterized by the formation of tumours on plant roots was observed in recently planted vineyards of the Meknes region (Morocco). The objective of this research was to analyse the diversity of pathogenic agrobacteria isolated from grapevine in Morocco. Eighty-two isolates from 11 affected vineyards were characterized by recA sequencing and were found to belong to Agrobacterium tumefaciens genomospecies G1, G4 or G7, Rhizobium rhizogenes, and to Allorhizobium vitis. Only the All. vitis isolates appeared to be pathogenic on tomato and multilocus sequence analysis phylogenetic analyses revealed a weak genetic diversity, with the definition of only four genomic groups. Definition of the All. vitis genomic groups correlated with specific pathogenic traits: indeed, genomic groups differed with respect to the severity of hypersensitive response symptoms on tobacco leaves, the intensity of necrotic response on grapevine explants and opine profiles. Both vitopine and octopine were detected by UHPLC in tumours induced by isolates of three genomic groups, an opine signature scarcely ever reported. Allorhizobium vitis is the only causative agent of crown gall on grape in Morocco, pathogenic isolates can be separated into four genomic groups. This study of recently crown-gall-infested vineyards demonstrated that All. vitis is the only causative agent and revealed the presence of nonpathogenic Agrobacterium strain within tumours. Moreover, as the genetic diversity of the All. vitis isolates is relatively narrow, this study lays the basis for further analyses on the evolution of the disease, on the dissemination of the pTi and more globally on the fate of the different genomic groups in this newly colonized environment.

Streptococcus pseudopneumoniae, an opportunistic pathogen in patients with cystic fibrosis

The pathogenic power of Streptococcus pseudopneumoniae has been specified over years, particularly in case of chronic respiratory diseases; S. pseudopneumoniae isolation has however not been characterized before in CF patients. Identification of S. pseudopneumoniae remains challenging due to the high simila-rity level between species of the Streptococcus mitis group. Twenty CF patients with S. pseudopneumoniae were included. Isolates initially identified by phenotypic routine methods were subjected to both recA sequencing and amplification of S. pseudopneumoniae specific markers. Microbiological and clinical data were reviewed for patients with confirmed S. pseudopneumoniae. Thirteen isolates actually belong to S. pseudopneumoniae. S. pseudopneumoniae was associated with pulmonary exacerbation in 46% of the patients, either as the sole pathogen or as part of a polymicrobial infectious process. S. pseudopneumoniae has to be considered as an additional opportunistic pathogen in CF and additional studies are needed to increase knowledge of its epidemiology and clinical significance in CF.

Reclassification of Micrococcus aloeverae and Micrococcus yunnanensis as later heterotypic synonyms of Micrococcus luteus.

Micrococcus aloeverae,Micrococcus endophyticus, Micrococcus luteus and Micrococcus yunnanensis are phenotypically and genotypically closely related, and together comprise the M. luteus group. In this study, the taxonomic relationships among Micrococcus aloeverae, M. luteus and M. yunnanensis were re-evaluated by using polyphasic approaches. The similarity values of the concatenated housekeeping gene (gyrB, recA and rpoB) sequences shared by the type strains of M. aloeverae, M. luteus and M. yunnanensis ranged from 98.3 to 99.4 %. The average nucleotide identity, average amino acid identity and digital DNA‒DNA hybridization values among these three taxa were greater (97.1‒98.1 %, 96.8‒98.1 % and 75.0‒83.5 %, respectively) than the thresholds for bacterial species delineation, indicating that they belong to the same species, whereas those for M. endophyticus were clearly lower than the thresholds. In addition, phenotypic and chemotaxonomic characterization results also support the synonymy of these three taxa. Therefore, we propose that M. aloeverae and M. yunnanensis should be reclassified as later heterotypic synonyms of M. luteus.

Ensifer fredii symbiovar vachelliae nodulates endemic Vachellia gummifera in semiarid Moroccan areas

The purpose of this work was to study the genetic diversity of the nodule-forming bacteria associated with native populations of Vachellia gummifera growing wild in Morocco. The nearly complete 16S rRNA gene sequences from three selected strains, following ARDRA and REP-PCR results, revealed they were members of the genus Ensifer and the sequencing of the housekeeping genes recA, gyrB, dnaK and rpoB, and their concatenated phylogenetic analysis, showed that the 3 strains belong to the species E. fredii. Based on the nodC and nodA phylogenies, the 3 strains clearly diverged from the type and other reference strains of E. fredii and formed a clearly separated cluster. The strains AGA1, AGA2 and AGB23 did not form nodules on Glycine max, Phaseolus vulgaris and Medicago truncatula, and effectively nodulated V. gummifera, Acacia cyanophylla, Prosopis chilensis and Leucaena leucocephala. Based on similarities of the nodC and nodA symbiotic genes and differences in the host range, the strains isolated from Moroccan endemic V. gummifera may form a different symbiovar within Ensifer species, for which the name “vachelliae” is proposed.

Elevation of Lactococcus lactis subsp. cremoris to the species level as Lactococcus cremoris sp. nov. and transfer of Lactococcus lactis subsp. tructae to Lactococcus cremoris as Lactococcus cremoris subsp. tructae comb. nov.

Currently, Lactococcus lactis contains four subspecies: L. lactis subsp. lactis, L. lactis subsp. hordniae, L. lactis subsp. cremoris and L. lactis subsp. tructae. In the study of Pérez et al., these four subspecies could be clearly divided into two groups based on recA sequence analysis: L. lactis subsp. lactis and L. lactis subsp. hordniae; L. lactis subsp. cremoris and L. lactis subsp. tructae. The two groups had a relatively low DNA-DNA hybridization value (about 60 %). In the present study, the taxonomic position of L. lactis subsp. cremoris and L. lactis subsp. tructae was re-examined based on sequence analyses of 16S rRNA, rpoB, recA and pheS genes, average nucleotide identity (ANI) values and digital DNA-DNA hybridization (dDDH) values. The result of 16S rRNA gene sequence analysis indicated that L. lactis subsp. cremoris NCDO 607T and L. lactis subsp. tructae L105T were phylogenetically related to the type strains of L. lactis subsp. hordniae, L. lactis subsp. lactis, Lactococcus taiwanensis, Lactococcus kimchii, Lactococcus allomyrinae, Lactococcus protaetiae, Lactococcus hircilactis, Lactococcus fujiensis and Lactococcus nasutitermitis. The 16S rRNA gene, rpoB, recA, pheS and concatenated rpoB, recA and pheS sequence similarities, ANI values, and dDDH values between the type strains of L. lactis subsp. cremoris, L. lactis subsp. tructae and phylogenetically related species were 93.5-99.4 %, 83.3-97.6 %, 80.6-92.4 %, 79.7-92.7 %, 83.5-94.3 %, 72.4-86.9 % and 21.4-32.5 %, respectively. Lower than 95-96 % ANI values and lower than 70 % dDDH values confirmed that the type strains of L. lactis subsp. cremoris and L. lactis subsp. tructae represent a novel species in the genus Lactococcus. Because L. lactis subsp. cremoris was proposed and validated before L. lactis subsp. tructae, L. lactis subsp. cremoris is elevated to the species level as Lactococcus cremoris sp. nov. and L. lactis subsp. tructae is transferred to L. cremoris as L. cremoris subsp. tructae comb. nov. The type strain of L. cremoris sp. nov. is NCDO 607T (=ATCC 19257T=DSM 20069T=JCM 16167T=LMG 6897T=NBRC 100676T). The type strain of L. cremoris subsp. tructae comb. nov. is L105T (=NBRC 110453T=DSM 21502T=JCM 31125T=LMG 24662T).

Yersinia kristensenii subsp. rochesterensis subsp. nov., isolated from human feces.

A single bacterial isolate, EPLC-04T, was isolated from human feces and identified as representing a member of the genus Yersinia on the basis of phenotypic characteristics, matrix assisted laser desorption ionization time-of-flight mass spectrometry and partial 16S rRNA gene sequencing. The isolate's phenotypic profile differed from that described for the most closely related species, Yersinia kristensenii, by exhibiting lipase production and lacking pyrazinamidase activity. Multiple genetic targets, including the complete (1465 bp) 16S rRNA gene sequence and partial sequences of groEL (539 bp), gyrB (935 bp), glnA (525 bp) and recA (535 bp) indicated that the isolate exhibited 98.91, 92.16, 90.81, 92.78 and 89.01 % identity with Yersinia aldovae, 98.98, 91.99, 90.17, 89.77 and 89.55 % identity with Yersinia intermedia, and 99.66, 98.11, 98.50, 98.49 and 98.51 % identity with Y. kristensenii, respectively. Phylogenetic reconstructions based on the combination of the four housekeeping genes indicated that the isolate formed a unique branch, supported by a bootstrap value of 100 %. Digital DNA-DNA homology and 16S rRNA gene sequencing identified EPLC-04T as representing Y. kristensenii. However, the unique phenotypic traits and results of phylogenetic analysis indicate that it represents a novel subspecies of Y. kristensenii. The name Yersinia kristenseniisubsp. rochesterensis subsp. nov. is proposed for this novel taxon (type strain EPLC-04T=ATCC BAA-2637T, DSMZ 28595T).

Mesorhizobium carbonis sp. nov., isolated from coal bed water.

A Gram-stain negative, aerobic, motile, short rod-shaped bacterium, designated as B2.3T, was isolated from coal bed water collected from Jincheng, Shanxi Province, China. The strain was able to grow at 10-40°C (optimum 28-30°C), pH 4.0-10.0 (optimum 7.0), and in the presence of 0-5.0% NaCl (optimum 3.0%, w/v). Phylogenetic analysis based on the 16S rRNA and concatenated housekeeping gene recA, atpD and glnA sequences showed strain B2.3T belongs to the genus Mesorhizobium, with Mesorhizobium oceanicum B7T as the closely related type strain. Strain B2.3T exhibited ANI value of 77.5% and GGDC value of 21.5% to M. oceanicum B7T. The major fatty acids were identified as summed feature 8 (C18:1ω7c and/or C18:1ω6c) and 11-methyl C18:1ω7c. The major polar lipids were found to consist of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine and an unidentified aminophospholipid. The predominant ubiquinone was identified as Quinone 10. Phenotypic and biochemical analysis results indicated that strain B2.3T can be distinguished from closely related type strains. On the basis of phenotypic, genotypic and chemotaxonomic characteristics, strain B2.3T is concluded to represent a novel species in the genus Mesorhizobium, for which the name Mesorhizobium carbonis sp. nov. is proposed. The type strain is B2.3T (=CGMCC 1.15730T = KCTC 52461T).

First Report of Pectobacterium parmentieri Causing Stem Rot Disease of Potato in Russia

Bacterial (aerial) stem rot of potato (Solanum tuberosum) in Russia is caused by the pectolytic bacteria Pectobacterium carotovorum subsp. carotovorum, P. atrosepticum, or Dickeya spp. (De Boer and Kelman 2001). In 2011 to 2014, over 100 pectinolytic enterobacterial isolates were obtained from green potato plants with blackleg symptoms found in four districts (Odintsovo, Dmitrov, Kashira, and Ruza) of Moscow region in severely infected commercial fields planted with cultivars Nevskii, Gala, and Lady Clear. Disease incidence was from 1 to 10% of infected plants in the field in July to August. From 5 to 15 samples were assayed for each of eight evaluated fields. Six bacterial strains isolated during the survey exhibited pectolytic ability on crystal violet pectate (CVP) agar and potato slices, and failed to grow at 37°C, but physiological tests did not conclusively distinguish the bacterium as P. atrosepticum (Khayi et al. 2016). They were identified as Pectobacterium parmentieri (formerly P. wasabiae group isolated from potato plants) (Kornev et al. 2012) based on their inability to elicit hypersensitive reaction on Nicotiana tabacum and their ability to utilize raffinose and lactose. These bacterial strains were gram negative, rod shaped, N-acetylglucosaminyl transferase, gelatin liquefaction, and ONPG-positive and positive for acid production from d-galactose, lactose, melibiose, raffinose, citrate, and trehalose. All strains were negative for indole production, phosphatase activity, reducing sucrose, and negative for acid production from maltose, sorbitol, inositol, inolin, melezitose, α-methyl-d-glucoside, and d-arabitol. All the strains exhibited pectolytic activity on potato slices. Reference strains of P. carotovorum NCPPB 312, P. atrosepticum NCPPB 549, and P. parmentieri LMG 29774 obtained from international collections were used as controls. Molecular identification of the bacterium was performed with 16S rRNA and recombinase A (recA) coding sequences as previously described (Waleron et al. 2002). BLAST analysis of the 16S rRNA DNA sequence for the strain F148 (syn. PB20) (GenBank accession no. MG493237.1) revealed 99% identity to the 16S rRNA of P. parmentieri WPP163 (Kornev et al. 2012). The other five strains had sequence of 16S rRNA identical to F148. Phylogenetic reconstruction using DNA sequences of recA gene from P. parmentieri F009, F034, F035, F127, F148 (PB20), and F149 (GenBank accession nos. MG518488, MG518489, MG518490, MG518491, and MG518492) and other related taxa clustered these bacteria with strain SCRI488 and other P. parmentieri strains, readily distinguishing them from other closely related species of Pectobacterium. Pathogenicity assays were conducted on stems of four young potato plants for each strain. Five-week-old ‘Docker’ potato plants were wound inoculated by injecting 10 μl of a bacterial suspension (10⁸ CFU/ml) into each stem. Plants were exposed to a 72-h incubation period with 90 to 100% relative humidity at 30°C, and lesions were measured. All inoculated plants exhibited stem rot/blackleg symptoms similar to those observed in the field, including brown water-soaked lesions. Symptoms were not observed on water-inoculated controls. The bacteria reisolated from all inoculated stems caused pitting on CVP and exhibited the same morphology as the original culture and, thus, were confirmed as P. parmentieri using 16S rRNA and recA sequences, fulfilling Koch’s postulates. To our knowledge, this is the first report of bacterial stem rot/blackleg of potato caused by P. parmentieri in the Russian Federation. According to results of several independent diagnostic laboratories, P. parmentieri had second place in occurrence on seed potatoes after P. carotovorum subsp. brasiliense (about 20% of the tested seed stocks) in field season 2017.

First Report of Pectobacterium polaris Causing Soft Rot of Potato in Poland

HomePlant DiseaseVol. 103, No. 1First Report of Pectobacterium polaris Causing Soft Rot of Potato in Poland PreviousNext DISEASE NOTES OPENOpen Access licenseFirst Report of Pectobacterium polaris Causing Soft Rot of Potato in PolandM. Waleron, A. Misztak, J. Jońca, and K. WaleronM. Waleron†Corresponding author: M. Waleron; E-mail: E-mail Address: [email protected]http://orcid.org/0000-0001-6789-2881Search for more papers by this author, A. MisztakSearch for more papers by this author, J. JońcaSearch for more papers by this author, and K. WaleronSearch for more papers by this authorAffiliationsAuthors and Affiliations M. Waleron † A. Misztak , Department of Biotechnology, Intercollegiate Faculty of Biotechnology, University of Gdansk and Medical University of Gdansk, 80-307 Gdansk, Poland J. Jońca K. Waleron , Department of Pharmaceutical Microbiology, Faculty of Pharmacy, Medical University of Gdansk, 80-416 Gdansk, Poland. Published Online:26 Oct 2018https://doi.org/10.1094/PDIS-05-18-0861-PDNAboutSectionsSupplemental ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat Bacteria belonging to the genus Pectobacterium are causal agents of soft rot disease all over the world, resulting in severe economic losses (Toth et al. 2011). In Poland, P. atrosepticum, P. parmentieri, P. carotovorum subsp. carotovorum, P. carotovorum subsp. odoriferum, and P. carotovorum subsp. brasiliense have been isolated from symptomatic plants to date (Motyka et al. 2017). In 2017, P. carotovorum strains were reclassified, and a new species, Pectobacterium polaris, was described (Dees et al. 2017). Therefore, we tested over 250 isolates that had been collected from plants with soft rot symptoms since 1995 in Poland to determine if P. polaris was present. A multilocus sequence analysis (MLSA) based on five housekeeping genes (gyrA, recA, recN, rpoA, and rpoS) (Waleron et al. 2018) was utilized to taxonomically characterize isolates. Based on our results, we found that the five strains isolated from potato (IFB5220, IFB5222, IFB5225, IFB5226, and IFB5252), and one from bittersweet (IFB5223) previously identified as P. carotovorum subsp. carotovorum (Waleron et al. 2002), should be renamed as P. polaris. All isolates were gram negative, facultative anaerobes exhibiting pectinolytic activity and were negative for oxidase, urease, indole production, gelatin liquefaction, and acid production from d-arabitol, dulcitol, sorbitol, raffinose, and melibiose. All strains were unable to utilize malonate and citrate. They were catalase positive, produced acid from lactose, rhamnose, saccharose, xylose, and trehalose, and were tolerant to 5% NaCl. All strains exhibited enzyme activity of cellulase, protease, α-amylase, α- and β-glucosidase, as well as ornithine and lysine decarboxylase. All strains except for IFB5252 and IFB5225 grew at 37°C. Strain IFB5226 produced reducing substances from sucrose and acid from maltose, utilized α-methyl-d-glucoside. All strains isolated from potato and the strain isolated from bittersweet caused soft rot symptoms on potato tuber tissues. The mean diameter of rotting potato tuber tissue (12 mm) was measured after incubation in 28°C and 95% relative humidity for 72 h. Surface-sterilized potato tubers were inoculated by inserting a pipette tip containing 25 μl of bacterial suspension (2 × 107 CFU/ml) 10 mm into each tuber. Three tubers were inoculated for each strain, and the inoculation was done in triplicate. As a control, sterile water was used. Sequence analysis of the partial 16S rRNA gene (1,435 bp) (MH166801 to MH166803, KU510098, KU510098, KU510101), amplified with primer pair 27F/1492R, of six aforementioned strains indicated that all of them (sharing identity from 98.36 to 100% between each other) exhibited 99 to 100% identity with P. polaris, strains NIBIO1006T and NIBIO1392 (GenBank accession nos. CP017481 and CP017482). The MLSA was performed on concatenated sequences of gyrA (KU510197, MH367256 to MH367259), recA (KU510110 to KU510114, MH367255), recN (KU510191, KU510192, KU510194, MH367267 to MH367269), rpoA (KU510163, KU510164, MH367263 to MH367266), and rpoS genes (KU510141, KU510143, MH367266 to MH367262). A consensus tree, which was constructed using the maximum likelihood method, clustered strains IFB5220, IFB5222, IFB5223, IFB5225, IFB5226, and IFB5252 with P. polaris strains available in GenBank. To our knowledge, this is the first report of P. polaris causing soft rot on potato in Poland. Based on the original isolation date, we can state that this species was present in Poland as early as 1996.

Phenotypic and Molecular Characterization of Brucella microti-Like Bacteria From a Domestic Marsh Frog (Pelophylax ridibundus).

Several Brucella isolates have been described in wild-caught and “exotic” amphibians from various continents and identified as B. inopinata-like strains. On the basis of epidemiological investigations conducted in June 2017 in France in a farm producing domestic frogs (Pelophylax ridibundus) for human consumption of frog's legs, potentially pathogenic bacteria were isolated from adults showing lesions (joint and subcutaneous abscesses). The bacteria were initially misidentified as Ochrobactrum anthropi using a commercial identification system, prior to being identified as Brucella spp. by MALDI-TOF assay. Classical phenotypic identification confirmed the Brucella genus, but did not make it possible to conclude unequivocally on species determination. Conventional and innovative bacteriological and molecular methods concluded that the investigated strain was very close to B. microti species, and not B. inopinata-like strains, as expected. The methods included growth kinetic, antimicrobial susceptibility testing, RT-PCR, Bruce-Ladder, Suis-Ladder, RFLP-PCR, AMOS-ERY, MLVA-16, the ectoine system, 16S rRNA and recA sequence analyses, the LPS pattern, in silico MLST-21, comparative whole-genome analyses (including average nucleotide identity ANI and whole-genome SNP analysis) and HRM-PCR assays. Minor polyphasic discrepancies, especially phage lysis and A-dominant agglutination patterns, as well as, small molecular divergences suggest the investigated strain should be considered a B. microti-like strain, raising concerns about its environmental persistence and unknown animal pathogenic and zoonotic potential as for other B. microti strains described to date.

Ignatzschineria cameli sp. nov., isolated from necrotic foot tissue of dromedaries (Camelus dromedarius) and associated maggots (Wohlfahrtia species) in Dubai.

Five bacterial strains, UAE-HKU57T, UAE-HKU58, UAE-HKU59, UAE-HKU60 and UAE-HKU61, were isolated in Dubai, UAE, from necrotic foot tissue samples of four dromedaries (Camelus dromedarius) and associated maggots (Wohrlfartia species). They were non-sporulating, Gram-negative, non-motile bacilli. They grew well under aerobic conditions at 37 °C, but not anaerobically. The pH range for growth was pH 7.0-9.0 (optimum, pH 7.5-8.0) and the strains could tolerate NaCl concentrations (w/v) up to 2 % (optimum, 0.5 %). They were catalase- and cytochrome oxidase-positive, but caseinase-, gelatinase- and urease-negative. Their phenotypic characters were distinguishable from other closely related species. Phylogenetic analyses of the almost-complete 16S rRNA gene and partial 23S rRNA gene, gyrB, groEL and recA sequences revealed that the five isolates were most closely related to undescribed Ignatzschineria strain F8392 and Ignatzschineria indica, but in most phylogenies clustered separately from these close relatives. Average nucleotide identity analysis showed that genomes of the five isolates (2.47-2.52 Mb, G+C content 41.71-41.86 mol%) were 98.00-99.97% similar to each other, but ≤87.18 % similar to other Ignatzschineriaspecies/strains. Low DNA relatedness between the five isolates to other Ignatzschineriaspecies/strains was also supported by Genome-to-Genome Distance Calculator analysis. The chemotaxonomic traits of the five strains were highly similar. They were non-susceptible (intermediate or resistant) to tetracycline and resistant to trimethoprim/sulphamethoxazole. The name Ignatzschineria cameli sp. nov. is proposed to accommodate these five strains, with strain UAE-HKU57T (=CCOS1165T=NBRC 113042T) as the type strain.

Marinobacter profundi sp. nov., a slightly halophilic bacterium isolated from a deep-sea sediment sample of the New Britain Trench

A piezotolerant, cold-adapted, slightly halophilic bacterium, designated strain PWS21T, was isolated from a deep-sea sediment sample collected from the New Britain Trench. Cells were observed to be Gram-stain negative, rod-shaped, oxidase- and catalase-positive. Growth of the strain was observed at 4-45°C (optimum 37°C), at pH 5.0-9.0 (optimum 7.0) and in 0.5-20% (w/v) NaCl (optimum 3-4%). The optimum pressure for growth was 0.1MPa (megapascal) with tolerance up to 70MPa. 16S rRNA gene sequence analysis showed that strain PWS21T is closely related to Marinobacter guineae M3BT (98.4%) and Marinobacter lipolyticus SM19T (98.2%). Multilocus sequence analysis (MLSA) based on sequences of housekeeping genes gyrB, recA, atpD, rpoB and rpoD indicates that strain PWS21T represents a distinct evolutionary lineage within the genus Marinobacter. Furthermore, strain PWS21T showed low ANI and diDDH values to the closely related species. The principal fatty acids were identified as C12:0, C12:0 3-OH, C16:1ω9c, C16:0 and C18:1ω9c. Ubiquinone-9 was identified as the major respiratory quinone. The polar lipids were identified as phosphatidylethanolamine (PE), phosphatidylglycerol (PG), diphosphatidylglycerol (DPG), aminophospholipid (APL), two unidentified lipids and an unidentified phospholipid (PL). The G + C content of the genomic DNA was determined to be 60.3mol%. On the basis of phenotypic, chemotaxonomic and molecular data, we conclude that strain PWS21T represents a novel species of the genus Marinobacter, for which the name Marinobacter profundi sp. nov. is proposed (type strain PWS21T = KCTC 52990T = MCCC 1K03345T).

Description of a novel species of fast growing mycobacterium: Mycobacterium kyogaense sp. nov., a scotochromogenic strain received as Mycobacterium vaccae.

A polyphasic study was undertaken to determine the taxonomic status of a rapidly growing, scotochromogenic organism that had been received as Mycobacterium vaccaeNCTC 11659T. The organism was found to have chemotaxonomic and cultural properties in accord with its assignment to the genus Mycobacteriumand was distinguished from the type strain of Mycobacterium vaccaeand from other closely related reference strains on the basis of concatenated sequences of 16S rRNA, gyrB, hsp65, recA and rpoB genes. It was also distinguished from M. vaccaestrain DSM 43292T and from the type strain of Mycobacterium obuense, its nearest phylogenetic neighbour, on the basis of chemotaxonomic and phenotypic data and digital DNA -DNA relatedness values of 22.7 and 68.3 %, respectively. These datasets not only indicate that strain NCTC 11659T had been misclassified as M. vaccae but that it merits recognition as representing a novel species of the genus Mycobacterium. It is proposed that the organism be classified as Mycobacteriumkyogaense sp. nov.